Ghrelin is a bioactive peptide that induces food intake, body weight gain, and adiposity in rodents (Tschop M. et al, Nature 407:908-13 (2000); Wren A. M. et al., Diabetes 50:2540-47 (2001)). Acute administration of ghrelin induces food intake in healthy men and women (Wren A. M. et al., J. Clin. Endocrinol. Metab. 86:5992-95 (2001); Druce M. R. et al., Int. J. Obes. Relat. Metab. Disord 29:1130-36 (2005)) as well as in cancer patients with anorexia (Neary N. M. et al., J. Clin. Endocrinol. Metab. 89:2832-36 (2004)). Repeated administration of ghrelin increases lean body mass, body weight and food intake in cachectic patients with Chronic Obstructive Pulmonary Disease (COPD) (Nagaya N. et al., Chest 128:1187-93 (2005)) and improved muscle wasting in patients with chronic heart failure (Nagaya N. et al, Circulation 110:3674-79 (2004)). A similar effect was also shown in a mouse model (Hanada T. et al., Biochem. Biophys. Res. Commun. 301:275-79 (2003)).
Tumor growth is associated with profound metabolic and neurochemical alterations, which can lead to the onset of the anorexia-cachexia syndrome. Anorexia is defined as the loss of the desire to eat, while cachexia results from progressive wasting of skeletal muscle mass and to a lesser extent adipose tissue, occurring even before weight loss becomes apparent. Cancer anorexia-cachexia syndrome is highly prevalent among cancer patients, has a large impact on morbidity and mortality, and impinges on patient quality of life. However, its clinical relevance is frequently overlooked, and treatments are usually only attempted during advanced stages of the disease (Laviano A. et al, Nat. Clin. Pract. Oncol. 3:158-65 (2005)).
Ghrelin is secreted in the pre-meal situation starting 1-2 hours before the meal resulting in a sharp, short-lived surge in plasma levels of ghrelin before the meal and lasting a short while after initiation of the meal. Since ghrelin is the only known peripherally-produced orexigenic (appetite-promoting) substance, it is believed that the increase in plasma levels of ghrelin is crucial for the initiation of the meal.
In its role as a key initiator of appetite, ghrelin, released from the endocrine cells in the mucosa of the gastrointestinal (GI) tract, may act both locally as a paracrine substance and centrally as a hormone, as discussed infra in the section related to cancer cachexia.
The GHRL (ghrelin) gene encodes a variety of products resulting from alternatively spliced transcripts, various types of cleavage of the prepropeptide, and various post-translational modifications (Kojima M. & Kangawa M., Physiol. Rev. 85:495-522 (2005); Zhang J. V. et al., Science 310:996-99 (2005)). In addition, different degradation products are produced by various tissues (De Vriese C. et al, Endocrinology 145:4997-5005 (2004)). Some of these GHRL products are described herein.
Ghrelin is a 28 amino acid peptide bearing an n-octanoyl side chain on the third serine, resulting from the cleavage of signal and propeptide from the 117 amino acid preproghrelin and an acylation event. The acylated N-terminus of ghrelin is essential for the endocrine functions (Kojima M. et al, Nature 402:656-60 (1999); Bednarek M. A. et al., J. Med. Chem. 43:4370-76 (2000)). Des-acyl ghrelin, which lacks the endocrine functions, was shown to have an antagonistic effect to that of ghrelin on glucose output in vitro (Gauna C. et al., J. Clin. Endocrinol. Metab. 89:5035-42 (2004)). An alternatively-spliced ghrelin mRNA encodes a 116 amino acid prepropeptide that is further processed to a Des-Gln14-ghrelin and a 27 amino acid processed peptide (Hosoda H. et al, J. Biol. Chem. 275:21995-22000 (2000)). Another peptide, Obestatin, is cleaved from the preproghrelin and has no sequence overlap with processed ghrelin peptide. This peptide was shown to have some antagonistic effect to acylated ghrelin, inhibiting food intake and body weight gain (Zhang J. V. et al., Science 310:996-99 (2005)). Yet another peptide, the 66 amino acid C-terminus of the preproghrelin, may also be functional (Pemberton C. et al, Biochem. Biophys. Res. Comm. 310:567-73 (2003)). A variety of isoforms, including isoforms encoded by different splice variants, are known for other proteins, e.g. for vascular endothelial growth factor (VEGF), where different isoforms share roles as angiogenesis, while differing in some other characteristics, such as binding affinity (Neufeld G. et al, FASEB J. 13:9-22 1999). Thus, the variety of products of the GHRL gene may reflect a similarly complex control of the endocrine and paracrine action of the ghrelin isoforms.
Previously, administration of ghrelin by continuous infusions of 5 pmol/kg/min doses for 270 minutes was shown to increase food intake in healthy humans (Wren A. M. et al., J. Clin. Endocrinol. Metab. 86:5992-95 (2001)). It was also shown that infusion of ghrelin for 90 minutes could increase food intake by 30% in cancer cachexia patients (Abstract P09, Digestive Hormones, Appetite and Energy Balance, Baylis and Starling meeting, London, June 2003). Recently, it was shown that subcutaneous injection of 3.6 nmol/kg acylated ghrelin prior to a meal, thereby ensuring a close mimic of the natural pre-meal situation, increased energy intake by 27%. Ghrelin also appeared to enhance the perceived palatability of the food offered (Druce M. R. et al, Int. J. Obes. Relat. Metab. Disord. 29:1130-36 (2005)).
These studies demonstrate that parenteral administration of ghrelin can increase appetite in both normal subjects and in patients with loss of appetite. Furthermore, Applicant has found that it is possible to obtain a significant effect of body weight gain and a significant increase in food consumption with a novel ghrelin splice variant (see co-owned, co-pending published U.S. Patent Application No. 2005/0059015, incorporated herein by reference) when administered to a subject, in particular when administered subcutaneously prior to a meal, thereby ensuring a close mimic of the natural pre-meal situation. Applicant's novel ghrelin splice variant effect of weight gain is mainly on lean mass while ghrelin's effect on weight gain is mainly on fat mass.